Project Summary The NIAID has made one of its highest priorities the development of a universal influenza treatment. Current therapeutic strategies for timely neutralization of influenza A virus (IAV), the causative agent of both seasonal and pandemic flu, have been minimally effective. Moreover, seasonal flu vaccine strategies are typically less than 50% effective. Therefore, there continues to be an urgent need to generate both novel vaccine and therapeutic strategies against IAV to prevent and combat large scale outbreaks. We propose to advance our product, CM-BT1, through proof-of-concept mouse studies that will demonstrate rapid neutralization of IAV. CM-BT1 is a self-amplifying RNA replicon capable of driving host cell production of a protein, specifically a broadly neutralizing anti- HA stalk antibody. CM-BT1 is a member of our proprietary library of replicons, modified RNA dependent RNA polymerases (RdRp) derived from a diverse collection of plant and invertebrate viruses. CM-BT1, our lead product, stems from turnip crinkle virus (TCV) and has been demonstrated to successfully generate an RNA capable of self-replication and protein production in healthy mouse lungs in vivo. Importantly, we have also demonstrated safety in that CM-BT1 can be turned off when engineered with additional open reading frames and a self-targeting cassette that enables complete control of replicon activity without inducing any overt cytopathic effects to cells. The use of RNA as a therapeutic platform is an exciting new area of therapeutics made possible by recent advances in chemistry and synthetic biology. However, this potential therapeutic platform still remains somewhat limited in scope, owing largely to the short- lived nature of RNA. Our platform technology, and lead product, CM-BT1, overcomes these limitations through ongoing and controlled production of relevant gene products that can function for days, weeks, or possibly months. The purpose of this proposal is to demonstrate proof-of-concept in vivo using two lethal challenge IAV models. Using go/no-go criteria, CM-BT1 will be advanced through rigorous product development culminating in an efficacy study that will enable the launch of this product into full scale preclinical development for timely therapeutic neutralization of IAV.